Genomic characterization of a virulent multidrug-resistant Salmonella enterica serovar Enteritidis strain isolated from meat rabbits (Oryctolagus cuniculus) in China

Abstract

Background

Salmonella enterica serovar Enteritidis (SE) is a major foodborne pathogen. While poultry is a known source, the threat from other livestock like rabbits (Oryctolagus cuniculus) is not well studied. This work investigates a highly virulent, multidrug-resistant (MDR) SE isolate, SE JL228, from a lethal rabbit farm outbreak in China in 2018. The objective of this study was to elucidate the genetic characteristics, antimicrobial resistance determinants, metal tolerance mechanisms, and virulence potential of a rabbit-derived isolate. This study provides important insights that advance our understanding of public and animal health risks.

Results

This isolate exhibited resistance to 13 antibiotics within 8 antimicrobial categories as well as to silver (Ag⁺), copper (Cu²⁺), and tellurium (Te⁴⁺). Additionally, SE JL228 demonstrated high tolerance to the quaternary ammonium compound (QAC) disinfectant. In vitro, it strongly invaded human brain endothelial cells (hBMECs) and survived longer inside macrophages than the moderately virulent SE strain LN248. In vivo studies confirmed extensive dissemination in mice, with an LD₅₀ approximately 68-fold lower than that of LN248. A 4.7-megabase chromosome together with two plasmids—pSE228A (211.4 kb, IncFIB/IncHI2) and pSE228B (54.6 kb, IncN)—was identified through whole-genome sequencing. The genome encoded 17 antibiotic resistance genes (ARGs), 34 virulence factors, heavy metal resistance operons (copESDBAC, silPABFCRE, terEDCBAZWYX), and QAC-resistance gene (qacE) on pSE228A. The plasmid pSE228A shares near-identical structure with a previously-isolated IncHI2 plasmid. The plasmid pSE228B harbors the transferable blaTEM-1B gene responsible for penicillin resistance.

Conclusions

The rabbit-derived SE JL228 represents a highly virulent, MDR, and metal-tolerant pathogen. Its ability to invade hBMECs, survive within macrophages, and disseminate systemically underscores its zoonotic potential. Transferable plasmids combining resistance and virulence determinants suggest an increased risk of dissemination within farm environments and beyond. These findings emphasize the urgent need for enhanced surveillance, prudent antimicrobial use, and effective biosecurity measures to mitigate the emergence and dissemination of such hybrid pathogens.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-025-04612-1.

Introduction

Salmonella enterica serovar Enteritidis (SE) represents a major non-typhoidal Salmonella (NTS) serovar associated with foodborne gastroenteritis in people. Globally, non-typhoidal Salmonella infections causes an estimated 93.8 million illnesses and 155,000 deaths worldwide each year [1]. Poultry meat, other types of meat, and eggs are the principal vehicles of transmission, posing major public health concerns worldwide [2]. Despite the abundant studies on SE in poultry, swine, cattle, and humans, little is known about its role in rabbit infections [3]. Salmonella infection in rabbits tends to develop acute or peracute septicemia, resulting in high morbidity and mortality [4]. China is the world's largest producer and consumer of rabbit meat, accounting for a significant portion of global production [5]. This large scale of production and consumption means that rabbit meat can be a significant vehicle for foodborne pathogens, making contamination a direct public health concern. These infections lead to major economic losses in commercial rabbit farming and concern about zoonotic transmission since rabbits are bred for their meat, fur, and as pets [6, 7].

Over the past few years, multidrug-resistant (MDR) SE have increasingly been reported, complicating therapeutic strategies due to resistance against multiple classes of antimicrobials [8]. Horizontal gene transfer (HGT) plays a central role in the dissemination of antimicrobial resistance (AMR) genes. It is mainly mediated by mobile genetic elements (MGEs), including plasmids, transposons, and integrons. As a result, MDR variants have emerged rapidly and been widely documented [9, 10]. Among these, plasmid-driven HGT is recognized as a key mechanism enabling AMR gene exchange among Enterobacteriaceae [11–13]. Among MDR SE strains, plasmids belonging to incompatibility groups such as IncF, IncHI, IncI, IncA/C, IncP and IncN have been widely implicated in the spread of resistance determinants [14]. Notably, these plasmids frequently co-carry both AMR and virulence genes, enhancing bacterial adaptability and pathogenicity [15].

Besides antibiotic resistance, bacteria adapting to heavy metals like copper (Cu²⁺), silver (Ag⁺), and zinc (Zn²⁺) has become an important factor in helping MDR pathogens continue to thrive and spread. These metals are commonly used as antimicrobial agents in animal husbandry, industrial settings, and medical applications [16, 17]. However, persistent exposure exerts selective pressure, promoting the emergence of bacterial strains that harbor metal resistance genes (MRGs). These genes are frequently co-located with AMR genes on plasmids. This co-selection mechanism enhances bacterial survival under heavy metal stress, further driving the spread of MDR strains in livestock and food production systems [18].

In this study, we characterized a SE isolate, JL228, obtained from an outbreak at a commercial meat rabbit farm in Jilin, China. The objective of this study was to elucidate the genetic characteristics, antimicrobial resistance determinants, and virulence potential of this rabbit-derived strain. To achieve this, we employed a comprehensive approach combining whole-genome sequencing (WGS) with in vitro cell-based assays and in vivo mouse models to fully characterize its pathogenic and zoonotic potential.

Material and method

Outbreak description

In March 2018, a veterinarian identified an outbreak of infectious disease at a commercial rabbit farm in Nong’an County, Jilin Province, China. The farm reared New Zealand White (NZW) rabbits (Oryctolagus cuniculus) for meat intended for the human food chain. More than 350 kits were affected, presenting with diarrhea, weight loss, and respiratory difficulty, leading to death in 68.5% of cases. Necropsies showed variable lesions, including hemorrhagic enteritis and splenomegaly. The most common finding was multifocal white lesions on the hepatic surface and parenchyma, corresponding histologically to focal necrotic hepatitis characterized by hepatocellular necrosis and infiltration of neutrophils and macrophages. These lesions are typical of septicemic Salmonella infection in rabbits.

Bacterial isolation and Serotyping

Liver samples from carcasses were aseptically collected and delivered to the laboratory for pathogen detection. Eight presumptive non-typhoidal Salmonella spp. Isolates were recovered from ten liver samples in Xylose lysine tergitol 4 (XLT4) agars with black colonies. They were then confirmed by VITEK®2 Compact microbial identification system (bioMérieux, NC, USA) and designated “JL227” to “JL234”. These isolates were classified as serovar Enteritidis using the White-Kauffmann-Le Minor scheme with standard agglutination methods and Salmonella antisera pools (S&A Reagents Lab, Bangkok, Thailand) [19]. As all eight isolates (JL227–JL234) exhibited identical serotypes and antimicrobial resistance profiles, isolate SE JL228 was selected as a representative strain for subsequent analyses, including whole-genome sequencing and in vivo experiments.

Antimicrobial, disinfectant, and heavy metal susceptibility

SE isolates were tested with VITEK®2 Compact system (bioMérieux, NC, USA) using VITEK®2 AST-GN65 card. Global-based Clinical and Laboratory Standards Institute (CLSI) guideline (2014–701) was applied for minimal inhibitory concentrations (MICs) interpretation. Susceptibilities of the strain SE JL228 to heavy metals were determined as described before [20]. These heavy metals including silver (AgNO₃, CAS#: 7761–88-8), copper (CuCl₂·2H₂O, CAS#:10,125–13-0), tellurium (K₂TeO₃, CAS#: 7790–58-1), cobalt (CoCl₂, CAS#: 7646–79-9), zinc (ZnSO₄, CAS#: 7733–02-0), chromium (CrCl₃·6H₂O, CAS#: 10,060–12-5), cadmium (Cd, CdCl₂, CAS#: 10,108–64-2) (Macklin Biochemical, Shanghai, China). In addition, the benzyldodecyldimethylammonium bromide (DBAB, CAS#: 7281–04-1) was chosen to evaluate resistance to the quaternary ammonium compound (QAC) disinfectant of SE JL228 as well. Escherichia coli (E. coli) ATCC™ 25,922 was chosen as a control strain.

Preparation of activated peritoneal macrophages (APMs)

BALB/c mice (6–8 weeks, Beijing HFK Bioscience) were housed in IVC at Ludong University with ad libitum food and water. Animals were handled according to the protocol reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ludong University (protocol No.: LDU-IACUC2019007). APMs isolation was performed according to previously described methods, with minor modifications [21]. Briefly, mice received 3% thioglycollate broth intraperitoneally 72 h before cell harvest. Prior to sampling, mice were anesthetized by inhalation of 4% isoflurane, followed by intraperitoneal injection of sodium pentobarbital at a dose of 150 mg/kg to induce euthanasia. Cervical dislocation was then performed as a secondary physical method to confirm death. After disinfection of the abdominal surface, 5 mL of ice-cold sterile phosphate-buffered saline (PBS) was injected intraperitoneally, and the abdomen was gently massaged. Subsequently, 4 mL of peritoneal lavage fluid was carefully aspirated and transferred into a sterile 15 mL conical tube placed on ice. The lavage fluid was centrifuged at 400 × g for 10 min at 4 °C to pellet the cells. The supernatant was discarded, and the cell pellet was resuspended in RPMI 1640 medium (Gibco, Suzhou, China) supplemented with 10% fetal bovine serum (FBS, ZETA, CA, USA). The peritoneal macrophages were cultured in RPMI 1640 with 10% FBS, washed with PBS after overnight incubation, and then stimulated with 1 µg/mL lipopolysaccharide (LPS; Sigma-Aldrich, MO, USA) for 24 h prior to performing the gentamicin protection invasion assay.

In vitro invasion assay

To evaluate bacterial invasion, gentamicin protection assays were performed using APMs and human brain microvascular endothelial cells (hBMECs) models, following established methods with minor modifications [22, 23]. Briefly, APMs or hBMECs were seeded into 24-well plates and cultured until a confluent monolayer was formed. JL 228 were grown to mid-logarithmic phase in LB broth, washed twice, and resuspended in pre-warmed cell culture medium. The cells were infected at a multiplicity of infection (MOI) of approximately 1:1 (bacteria to host cells) and incubated for 1 h at 37 °C in 5% CO₂. Following infection, the monolayers were washed three times with PBS to remove non-adherent bacteria and subsequently incubated for 1 h in medium containing gentamicin (100 µg/mL) to eliminate extracellular bacteria. The medium was then replaced with fresh medium supplemented with gentamicin (10 µg/mL) for maintenance. At 24 h post-infection, host cells were lysed with 0.1% Triton X-100, and intracellular bacteria were quantified by plating serial dilutions on XLT4 agar to determine colony-forming units (CFU). LN248, another SE strain with moderate invasive capability [24], and E. coli ATCC 25922™ were included as controls.

Pathogenicity in mice

SE JL228 and LN248 were cultured in LB broth and subcultured at 37 °C with shaking at 180 rpm for 2.5 h. Bacteria were washed with PBS and diluted to 2 × 10⁹ CFU/mL. Mice were inoculated intraperitoneally with 50 μL of serial dilutions of the bacterial suspension. Mortality was recorded over a 28-day observation period, and the median lethal dose (LD₅₀) was calculated using the Reed and Muench method [25]. For LD₅₀ and infection studies, mice were monitored at least two times daily and up to four times daily during the acute phase. Humane endpoints were predefined: severe respiratory distress, inability to eat/drink, severe neurologic signs, or moribund state. Animals meeting humane endpoint criteria were humanely euthanized immediately as described above. SE JL228 dissemination in mice was evaluated as a previously described [26]. Mice were fasted for 4 h before per os (p.o.) treatment with 20 mg streptomycin. After 20 h, they were intragastric infected with 1 × 10⁸ CFU of SE JL228 or SE LN248. Five days post-infection, mice were anesthetized and euthanized, and their livers, spleens, brains, and mesenteric lymph nodes were aseptically collected. Each organ was weighed and placed in sterile PBS (1 mL PBS per 0.1 g tissue) and homogenized individually using a mechanical tissue homogenizer until a uniform suspension was obtained. The resulting homogenates were serially diluted tenfold (10⁻¹ to 10⁻⁶) in sterile PBS, and 100 μL of each dilution was plated onto XLT4 agar plates to assess bacterial dissemination and colonization in the respective organs.

Whole-genome sequencing (WGS)

The genomic DNA of SE JL228 was extracted using an QIAamp DNA Mini Kit (Qiagen, MD, USA) according to the manufacturer’s instructions. The genome was resolved using the Unicycler (v0.4.8) hybrid assembly pipeline with long reads generated by the PacBio® sequencing platform (Pacific Biosciences, CA, USA), yielding an average sequencing coverage of approximately 100 ×, with SE strain P125109 (GenBank accession No. NC_011294) as the reference genome [27]. This genome assembly was further proofread using short PE reads generated by the Illumina® Hiseq × 10 (Illumina Inc., CA, USA) platform at a sequencing coverage of approximately 200 ×. Plasmid sequences were identified and recovered by PlasFlow software in the above metagenomic data [28], followed by gene annotation using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) software on PLSDB plasmid database [14]. Genomic and plasmid sequence analyses were performed using the online platform of Majorbio Cloud Platform (www.majorbio.com). Whole-genome including chromosome and plasmid sequences were deposited in the Genome database under accession No. CP094269 to CP094271 in the National Center for Biotechnology Information (NCBI).

Molecular typing and prediction

Chromosomal and plasmid sequences were analyzed using the Achtman multilocus sequence typing (MLST), core genome multilocus sequence typing (cgMLST), and replicon sequencing typing (RST) schemes available in the PubMLST database (http://pubmlst.org). Antibiotic resistance and virulence determinants were predicted via ResFinder 4.0 and VFDB, respectively [29, 30].

Genomic comparison visualization

Plasmid circular maps were created to compare a reference bacterial plasmid to all query bacterial strains using BLAST Ring Image Generator (BRIG). To search for similar metal resistance loci and genetic organization. The multi-heavy metal resistance region of plasmid pSE228A (range: 1–86,200 bp) was compared against the nucleotide database using blastn program (date: April 10th, 2022). Easyfig 2.2.5 software was used for creating linear comparison figures of multiple genomic loci [31].

Fig. 2.

Bacterial Dissemination and Colonization of SE JL228 and LN248 in Mice. A Survival curves of mice orally infected with graded doses of SE JL228 or SE LN248. B Body-weight changes of infected mice over 14 days. C–G Bacterial loads in liver, spleen, mesenteric lymph nodes, and brain at 5 days post-infection. Data are presented as log₁₀ CFU/gram (mean ± SD, n = 6). Data represent mean log of CFU per gram of tissue ± SD from three independent experiments (n = 6). Significance levels are denoted as ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test)

Plasmid conjugal transfer

Filter and liquid mating methods were employed to evaluate the conjugative ability of SE JL228, following previously described protocols with minor modifications [32, 33]. E.coli C600 as the recipient. For filter mating, donor and recipient cultures (1 mL each, grown to OD600 ≈ 0.8) were mixed, centrifuged, and the pellet was resuspended in 50 µL LB broth, spotted onto a 0.45 µm filter on an LB agar plate, and incubated at 37 °C for 18 h. For liquid mating, 1 mL of the donor culture and 1 mL of the recipient culture were mixed in 8 mL of LB broth and incubated statically at 37 °C for 18 h. After incubation, the cultures were collected, serially diluted, and plated on selective LB agar containing ampicillin (100 µg/mL) and rifampicin (200 µg/mL) to recover E. coli C600 transconjugants, which are intrinsically resistant to rifampicin. To verify plasmid transfer, colonies growing on selective plates were screened for the blaTEM-1B gene and the corresponding penicillin-resistance phenotype. The positivity rate was calculated as the number of confirmed transconjugants divided by the total number of colonies tested. The blaTEM-1B gene of the transconjugants was amplified using PrimeSTAR MAX DNA Polymerase (Takara, Beijing, China). Each 50-µL PCR reaction contained 25 µL of PrimeSTAR MAX DNA Polymerase, 3 µL of each primer (forward: 5′-ATGAGTATTCAACATTTCCGTG-3′; reverse: 5′-TTACCAATGCTTAATCAGTGAG-3′; 10 µM), 100 ng of template DNA, and ddH₂O to a final volume of 50 µL. The PCR amplification was carried out under the following conditions: initial denaturation at 98 °C for 1 min; 30 cycles of 98 °C for 10 s, 55 °C for 10 s, and 72 °C for 60 s; followed by a final extension at 72 °C for 2 min. Electrophoresis was performed on 1% agarose gels at 110 V for 30 min. After staining with GelRed, the DNA bands were visualized under UV illumination.

Statistical analysis

Data were analyzed using GraphPad Prism 9 (GraphPad Software, La Jolla, CA). Statistical significance between two groups was determined using an unpaired, two-tailed Student’s t-test. Significance levels are denoted as ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001.

Result

Isolate Identification and Serotyping

Of 10 liver samples, 8 tested positives for non-typhoidal Salmonella spp. via XLT4 agar isolation, showing black-centered colonies with yellow/pink periphery after 18 h, turning fully black by 30 h. All isolates matched Salmonella enterica serovar Enteritidis biochemical and antigenic profiles (9,12:g,m:-) based on VITEK 2 GN tests and serotyping (Additional file 1).

SE JL228 isolate exhibits multidrug resistant (MDR)

Antimicrobial resistance profiles were observed in all SE isolate, which exhibited complete resistance to 12 tested antibiotics (Table 1). MIC assays confirmed high-level tolerance to silver (Ag⁺, 32 mg/L), copper (Cu²⁺, 1024 mg/L), and tellurite (Te⁴⁺, 16 mg/L). The isolate also displayed a high MIC for the quaternary ammonium compound (QAC) disinfectant DBAB (51.2 mg/L), substantially exceeding control strains (Table 2).

Table 1.

Antimicrobial drug susceptibility profiles of all eight isolates (SE JL227-JL234)

antimicrobial	MIC (mg/L)	susceptibility interpretation
Penicillins
Ampicillin (AMP)	≥ 32	R
Amoxicillin/Clavulanic acid (AMC)	16	I
Piperacillin (PIP)	≥ 128	R
Cephalosporin
Cephalexin (CL)	≥ 64	R
Cefovecin (CEF)	≥ 8	R
Ceftiofur (EFT)	≥ 8	R
Cefpodoxime (CPD)	4	I
Carbapenems
Imipenem (IPM)	≤ 1	S
Aminoglycosides
Amikacin (AMK)	≥ 64	R
Gentamicin (GEN)	≥ 16	R
Tobramycin (TOB)	≥ 16	R
Sulphonamides
Trimethoprim/Sulfamethoxazole (SXT)	≥ 320	R
Fluoroquinolones
Marbofloxacin (MAR)	2	I
Enrofloxacin (ENR)	≥ 4	R
Chloramphenicol
Chloramphenicol (CHL)	≥ 64	R
Tetracycline
Tetracycline (TET)	≥ 16	R
Nitrofurantoin
Nitrofurantoin (NIT)	128	R

All eight isolates (JL227–JL234) exhibited identical resistance profiles

Antimicrobial categories are shown in bold

MIC minimum inhibitory concentration, S susceptible, R resistant, I intermediate

Table 2.

Minimum inhibitory concentrations to heavy metals and QAC^a of SE JL228

Strain/isolate	Ag+ (mg/L)	Cd2+(mg/L)	Co2+(mg/L)	Cr3+(mg/L)	Cu2+(mg/L)	Te4+ (mg/L)	Zn2+ (mg/L)	DBABb(mg/L)
SE JL228	32	128–256	256	512	1024	16	1024	51.2
SE LN248	8	128	256	512	256	4	1024	25.6
E. coli ATCC 25922™	8	128–256	64–128	512–1024	256	0.5–1	1024	25.6
MIC50c	10 ± 2	153 ± 46	268 ± 40	550 ± 21	256 ± 24	5 ± 0.7	1431 ± 201	ndd

^aQAC, quaternary ammonium compound

^bDBAB, benzyldodecyldimethylammonium bromide

^cMIC₅₀ shown here were determined from 113 SE isolates

^dnd, not determined

SE JL228 exhibits high virulence, invasiveness, and systemic dissemination

The virulence phenotype of SE JL228 was benchmarked against the moderate-virulence strain SE LN248. In vitro, SE JL228 demonstrated significantly higher invasion of hBMECs and superior intracellular survival within activated peritoneal macrophages (APMs) at 24 h post-infection (Fig. 1).

Fig. 1.

Invasion and intracellular survival of SE JL228 and LN248 in hBMECs and APMs. A Invasion efficiency of SE JL228 and LN248 in hBMECs and APMs. B Intracellular survival of both strains in hBMECs and APMs at 24 hpi determined by gentamicin protection assay. Data are expressed as CFU per well (mean ± SD, n = 10). Significance levels are denoted as ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test)

The isolate exhibited markedly higher pathogenicity in vivo. The oral LD₅₀ in BALB/c mice for SE JL228 was 5.0 × 10⁶ CFU, approximately 68-fold lower than that of LN248 (3.4 × 10⁸ CFU). In a streptomycin-pretreated mouse model, 100% of mice infected with 1 × 10⁸ CFU of SE JL228 died by day 14, contrasting with a 40% survival rate for the LN248 group (Fig. 2A). Infection with SE JL228 also correlated with more rapid weight loss (Fig. 2B) and the onset of neurological symptoms, including ataxia, in 50% of the cohort (Additional file 2). Consistent with these clinical signs, bacterial loads recovered from the liver, spleen, and brain were significantly higher in SE JL228-infected mice, confirming its enhanced capacity for systemic dissemination (Fig. 2C-E, 2G).

Genomic overview and molecular typing

The hybrid assembly, utilizing both PacBio long-read and Illumina short-read data, successfully generated a complete, circular chromosome (4.7 Mb, CP094269) and two complete, circular plasmids, pSE228A (211.4 kb, CP094270) and pSE228B (54.6 kb, CP094271). GC-content values were 52.17% for the chromosome, 46.28% for pSE228A, and 47.51% for pSE228B. Using NCBI’s PGAP annotation tool [34], a total of 4,896 genes were identified, with 4,612 on the chromosome, 223 on pSE228A, and 61 on pSE228B (Fig. 3). According to the Plasmid MLST typing schemes, plasmid pSE228A was identified as a multiple-replicon plasmid belonging to the incompatibility groups IncFIB and IncHI2 [35]. The same analysis indicated that plasmid pSE228B belongs to the IncN group.

Fig. 3.

Circular map of SE JL228 chromosome and plasmids. Chromosome SE JL228, plasmids pSE228A and pSE228B, are shown, with plasmids depicted independently of the chromosome scale. From outer to inner rings: protein-coding genes on the forward strand (colored by COG categories), forward-strand genes, reverse-strand genes, protein-coding genes on the reverse strand, G + C content, and G + C skew. The legend indicates plasmid backbone, accessory modules, virulence, metal resistance, and AMR genes with distinct colors

Structure of the plasmids carried by isolate SE JL228

Plasmid pSE228A contained 223 predicted genes, including two backbone repB loci located at positions 146,725–147,714 and 86,251–87,000 (complementary strand). Additional loci associated with IncHI-type conjugation and plasmid maintenance were also present, forming a repB-traE-traK-trhB-trhV-traC-parA-parM-htdF-rsp-trhF-trhU-trhN-trhI cluster (Fig. 4A). Furthermore, a second repB locus (86,251–87,000), together with MPK88_23790 (DNA replication terminus site-binding protein), ccdA, ccdB, trhH, and trhR, may contribute to plasmid replication, stability, and transfer (Fig. 4B).

Fig. 4.

Genetic organization of plasmids pSE228A and pSE228B in isolate SE JL228. A Genetic structure of the repB-traE-traK-trhB-trhV-traC-parA-parM-htdF-rsp-trhF-trhU-trhN-trhI loci in pSE228A, which are involved in plasmid replication, maintenance, and conjugation. In addition, two related genes encoding pili assembly chaperone and plasmid transfer protein, corresponding to gene MPK88_23540 and MPK88_23545 were also found between repB and traE. B Additional replication and stability-related genes in pSE228A. This section includes a second repB gene, along MPK88_23790, ccdA, ccdB, trhH, trhR. C The IncN-type plasmid pSE228B carries key replication (repM, MPK88_24365, MPK88_24370, MPK88_24425) and maintenance (parA, parG, MPK88_24355 (RelE/ParE toxin), MPK88_24360 (plasmid stabilization protein)) genes. Additionally, ddp3, encoding a DNA distortion polypeptide, is present

Plasmid pSE228B contained a backbone region spanning positions (30,357–43644 bp). This region encoded four replication initiation proteins, including repM and three uncharacterized replication-associated proteins (MPK88_24365, MPK88_24370, MPK88_24425). Additionally, four maintenance-related genes were identified: parA, parG, MPK88_24355 (RelE/ParE toxin), and MPK88_24360 (stabilization protein), along with ddp3, encoding a DNA distortion polypeptide.

Antimicrobial and heavy metal resistance genes

Using the ResFinder4.0 platform, seventeen antimicrobial resistance (AMR) genes were detected in the genome of SE isolate JL228. The chromosomal gene aac(6’)-Iaa was identified, along with three genes located on plasmid pSE228A [aac(6’)-IIc, sul1, and ere(A)], and ten genes carried on plasmid pSE228B [armA, aac(6’)-Ib-cr, aac(3)-IId, aph(6)-Id, aph(3’’)-Ib, aph(3’)-Ia, blaTEM-1B, mph(E), sul1, and sul2]. (Fig. 5A–C). These AMR genes in chromosome and plasmids are predicted to confer resistance to seven class antibiotics (Additional file 3). Besides, quaternary ammonium compound-resistance gene qacE were also found in both plasmids, with the predicted phenotype of Benzylkonium Chloride, Ethidium Bromide, Chlorhexidine and Cetylpyridinium Chloride resistance (Fig. 5B and C).

Fig. 5.

Distribution of antimicrobial resistance (AMR) genes in the chromosome and plasmids of JL228. A Chromosomal AMR gene. The aac(6 ‘)-Iaa gene, conferring resistance to aminoglycosides, is located within the chromosome. Flanking genes (gene2476 and rspA) are shown in gray, while the AMR gene is highlighted in cyan. B AMR gene cluster in plasmid pSE228A. A cluster of AMR genes, including sul1, ere(A) and aac(6')-IIc, is present within the pSE228A. This cluster is flanked by IS6-like transposase genes and contains a class 1 integron integrase gene (intI1), facilitating gene mobility. The presence of MPK88_24140, encoding an NAD (+)-rifampin ADP-ribosyltransferase, suggests potential rifampin resistance. C AMR genes in plasmid pSE228B. The 54.6-kb IncN-type plasmid pSE228B carries eleven AMR genes (armA, aac(6')-Ib-cr, aac(3)-IId, aph(6)-Id, aph(3'')-Ib, aph(3')-Ia, blaTEM-1B, mph(E), sul1, sul2, msr(E)), scattered across four regions. The qacE gene, conferring resistance to quaternary ammonium compounds, is also present. D Plasmid pSE228A harbors three heavy metal resistance operons: (Ⅰ) Copper resistance locus (copESDBAC, region 15,559–20,810)–Genes involved in copper resistance are shown in green. (Ⅱ) Silver resistance locus (silPABFCRE, region 22,094–34,545)–Genes responsible for silver resistance are depicted in white. (Ⅲ) Tellurite resistance locus (terEDCBAZWYX, region 62,141–75,496)–Tellurite resistance genes are marked in purple

Three heavy metal tolerance operons were identified on pSE228A: copper (copESDBAC), silver (silPABFCRE), and tellurite (terEDCBAZWYX) (Fig. 5D, Table 3). The large multi-metal resistance region of pSE228A is flanked by mobile genetic elements (MGEs) including IS26 family transposase, Tn5403 family transposase, IS100 family transposase istA and istB (Fig. 5D). Comparative genomics revealed the multi-metal resistance region on pSE228A was highly conserved with plasmids from diverse bacterial genera, including E. coli and S. Typhimurium (Fig. 6).

Table 3.

Molecular characteristics of plasmids carried by the SE JL228

plasmid	Size (kb)	ST	Inc group	Replication, plasmid conjugal transfer and maintenance gene	AMR gene and heavy metal resistance gene	Virulence plasmid-encoded traits
pSE228A	211.4	14	IncFIB-IncHI2	repB (MPK88_23535), traE, traK, trhB, trhV, traC, parA, parM, htdF, rsp, trhF, trhU, trhN, trhI, MPK88_23540, MPK88_23545, repB (MPK88_23820), MPK88_23790, ccdA, ccdB, trhH, trhR	copper resistance: copA, copB, copC, copD, copS, copE, MPK88_23180 silver resistance: silP MPK88_23200 silA, silB silF silC silSR silE tellurite resistance: terE, terC, terD, terB, terA, terZ, terW, terY, terX antibiotic resistance: aac(6')-IIc, sul1, ere(A)a QAC resistance: qacEb	spvDBAR: Salmonella plasmid virulence ibeBc: Invasion of brain endothelial cells
pSE228B	54.6	23	IncN	repM, MPK88_24365, MPK88_24370, MPK88_24425, parA, parG, MPK88_24360, MPK88_24360, ddp3	antibiotic resistance: armA, aac(6')-Ib-cr, aac(3)-IId, aph(6)-Id, aph(3'')-Ib, aph(3')-Ia, blaTEM-1B, mph(E), sul1, sul2 QAC resistance: qacEb	none

^aIncomplete, 85% coverage

^bIncomplete, 84.7% coverage

^csynonyms name, silC for this study, cusC, in E. coli

Fig. 6.

Plasmid Comparison Reveals Conserved Multi-Metal Resistance Locus in pSE228A. A The gene collinearity alignment of pSE228A with pKO_1 and XY-1 plasmid unnamed1. B The gene collinearity alignment of pSE228A with pEC5207 and pR15.0430. C The gene collinearity alignment of pSE228A with pYUSHP2-1. The multi-heavy metal resistance region of pSE228A (Red label, 1–86,200) was compared with the resistance region in other plasmids from different bacterial sources. (Green: copper resistance gene; Purple: tellurite resistance gene; Gray: silver resistance gene; Red: other). The plasmid information involved in the comparison is detailed in Additional file 7

Virulence factor (VF) genes

A total of 158 VFs were cataloged (Additional file 4). The chromosome possessed gene clusters for the SPI-1 and SPI-2 Type III Secretion Systems (T3SS), as well as key regulators (csgD, adrA) and structural operons (csg, bcs) for biofilm formation. The primary genetic basis for the hypervirulent phenotype was linked to plasmid pSE228A. This plasmid was found to harbor a complete Salmonella plasmid virulence (spv) operon (spvRABCD). This operon is a known determinant for systemic infection and intracellular proliferation within macrophages. Notably, pSE228A also carried the ibeB gene (annotated as silC/cusC in this study), an ortholog associated with the invasion of hBMECs in E.coli, providing a genetic correlate for the neurological symptoms observed. Its orthologous gene in Escherichia encoding a virulence factor involving in invasion of brain endothelial cells. The deduced amino acid sequence of SE JL228 shares approximate 71% identities (71.09%−71.33%) with its orthologs in pathogenic Escherichia. (Fig. 7). The chromosomal VF arsenal included multiple fimbrial operons (e.g., fim, lpf, stf), adherence factors (pagN, sadA), and macrophage survival genes (mgtC). In addition, a Peg fimbrial adherence determinant was found exclusively in LN248. (Additional file 5).

Fig. 7.

Conserved Amino Acid Alignment of JL228 and Orthologs from Pathogenic Escherichia coli. The sequences include ECP_0603_UPEC, EC55989_056_EAEC, O3K_18755_StxEAEC, APECO1_1476_APEC, Z0711_EHEC, and UMNK88_601_ETEC. Identical residues are highlighted, showing a high degree of sequence conservation. The alignment reveals approximately 71% sequence identity (70.72%–71.15%) between JL228 and its orthologs. Gaps indicate sequence variations among different strains

Comparative analysis of plasmids

The overall backbone of pSE228A was nearly identical to plasmid pSE_AH228 (GenBank accession ON960346.1) from another Salmonella isolate (Fig. 8A). pSE228B shared limited homology with known plasmids, though its AMR modules were conserved in IncN plasmids from K. pneumoniae and Salmonella (Fig. 8B).

Fig. 8.

Comparative plasmid maps for SE JL228 and related plasmids. A Comparison of plasmid pSE228A. From outer to inner circle: CP091471.2, CP073772, CP067067, CP055064, CP031284, AP028562, CP139029, CP115835, CP113164, MH399264, KT347600, MN423361, ON960346, OW849257, GC skew −, GC skew +, GC content. B Comparison of plasmid pSE228B. From outer to inner circle: CP041175, CP031234, CP029683, CP026570, CP026156, CP017233, CP096599, CP063509, GC skew −, GC skew +, GC content. Detailed plasmid information are provided in Additional file 8 and 9

Transferability assay

Verification of the transconjugants identified eight positive isolates. Membrane filter mating yielded three confirmed clones (11.1% positivity rate), while liquid mating yielded five (15.6% positivity rate). PCR confirmed the blaTEM-1B gene (850-bp amplicon) in all transconjugants, identical to SE JL228, with sequencing verifying blaTEM-1B presence (Fig. 9). Broth microdilution showed high-level penicillin resistance (MIC > 2048 μg/mL) in all transconjugants, consistent with SE JL228 and higher than E. coli C600 (Additional file 6).

Fig. 9.

Confirmation of plasmid conjugation and resistance gene transfer in transconjugants. A PCR amplification of the blaTEM-1B gene in all eight transconjugants. Lane M: DNA marker; Lane 1: negative control; Lane 2: E. coli C600; Lane 3: SE JL228 (positive control); Lanes 4–11: transconjugants-1–8. All transconjugants produced an 850 bp amplicon identical to the donor isolate SE JL228. B Nucleotide sequence alignment of the blaTEM-1B amplicon from transconjugants with the reference blaTEM-1B gene sequence. The alignment confirmed 100% sequence identity between the transconjugants and the donor isolate JL228, validating the horizontal transfer

Discussion

In this study, we characterize a SE isolate JL228, from a rabbit farm outbreak, revealing its high resistance to multiple antibiotics and metals mediated by plasmid and chromosomal determinants (Fig. 10). Notably, the MDR profile of SE JL228, encompassing resistance to penicillin, tetracyclines, sulfonamides, and fluoroquinolones, closely mirrors the antibiotic classes most commonly used in Chinese livestock production [36]. This strong correlation suggests that the strain’s resistome has been shaped by the selective pressures exerted by local veterinary practices. The observed clinical failure of tetracycline and penicillin, kanamycin treatments during the initial outbreak provides a clear illustration of this relationship, underscoring the significant challenges that such MDR strains pose to effective infection control in farm environments. In addition, the isolate also showed increased virulence, enhanced systemic dissemination, and the ability to cross the blood–brain barrier and invade hBMECs. This highly resistant and virulent isolate poses a significant threat to animal and human health, necessitating close monitoring in farms and hospitals.

Fig. 10.

Genetic architecture of virulence and resistance in SE JL228. This schematic illustrates the genomic architecture of the hypervirulent, MDR SE strain JL228, comprising a 4.7 Mb chromosome and two plasmids, pSE228A and pSE228B. Together, these elements define three major pathogenic mechanisms: (1) Synergistic intracellular survival and systemic dissemination: Core chromosomal virulence determinants, including Type III Secretion Systems (T3SS, SPI-1/SPI-2) and adherence-associated genes, form the foundation for host cell invasion. This is further enhanced by the 211.4 kb IncFIB/IncHI2 plasmid pSE228A, which encodes the complete Salmonella plasmid virulence (spvRABCD) operon—a key factor promoting systemic infection and macrophage survival. pSE228A also carries heavy metal resistance operons, providing a co-selective advantage under farm-related metal exposure. (2) Plasmid-mediated neuroinvasion: pSE228A harbors the ibeB gene, an ortholog implicated in the invasion of hBMECs, correlating with the neuroinvasive phenotype and neurological manifestations observed in infected mice. (3) Comprehensive antimicrobial resistance: The extensive resistome of JL228 is distributed across both plasmids. The 54.6 kb IncN plasmid pSE228B carries ten ARGs, including armA and blaTEM-1B, while pSE228A contributes additional ARGs, heavy metal resistance operons, and disinfectant resistance genes. Collectively, the 17 ARGs and two metal resistance operons confer high-level resistance to 13 antibiotics, quaternary ammonium compound (QAC) disinfectants, and multiple heavy metals, explaining the observed therapeutic failure of penicillin, tetracycline and kanamycin during the farm outbreak. Light green squares indicate multidrug resistance genes; orange squares represent virulence factor genes; and light blue squares denote heavy metal resistance genes

The SE JL228 genome consists of two plasmid pSE228A and pSE228B. Plasmid pSE228A, likely a fusion of IncFIB and IncHI2, encodes multiple ARGs, MRGs, and VFs, flanked by mobile genetic elements including IS6-like element IS26 family transposase, Tn3-like element Tn5403 family transposase, IS21-like element IS100 family transposase istA and istB. Plasmid pSE228A harbors both antimicrobial resistance and virulence genes, functioning as a high-risk functional gene reservoir that facilitates the dissemination of MDR through efficient HGT [37]. It contains operons conferring resistance to copper, silver, and tellurite, together with ARGs such as sul1, ere(A), and aac(6’)-IIc, indicating that composite resistance phenotypes are maintained under dual selective pressures from metals and antibiotics via a co-selection mechanism. The widespread use of copper as a feed additive in rabbit production may lead to persistent environmental metal stress. This stress can enable MDR SE strains to survive and spread even in the absence of antibiotic exposure [38, 39]. This process effectively decouples the persistence of ARGs from direct antibiotic use, forming a stable environmental resistome. Consistent with previous findings, SE isolates from intensive farms and wastewater treatment facilities frequently co-harbor MRGs and ARGs [40, 41], a phenomenon also observed in other Enterobacteriaceae such as Vibrio cholerae and Klebsiella pneumoniae [42–44]. Importantly, pSE228A is a conjugative IncFIB-IncHI2-type plasmid with high mobility, enabling the co-transfer of resistance, metal tolerance, and virulence determinants (e.g., spv and ibeB) as functional modules. Comparative genomic analysis revealed that pSE228A is highly homologous to Salmonella plasmid pSE_AH228 and shares conserved resistance structures with Klebsiella pneumoniae plasmids, suggesting potential interspecies horizontal transfer. In contrast, plasmid pSE228B, an IncN-type plasmid commonly found in Enterobacteriaceae, does not encode virulence factors but carries several ARGs, including armA and blaTEM-1B, which substantially contribute to the MDR phenotype of SE JL228 and complicate therapeutic interventions. Conjugation experiments confirmed that pSE228B can be efficiently transferred to other members of the Enterobacteriaceae via HGT, underscoring its critical role in the interspecies dissemination of antimicrobial resistance. Although our in vitro conjugation assays focused on this clinically important IncN plasmid—which itself lacks a complete conjugation machinery—genomic analysis revealed that pSE228A carries a full trh-associated transfer locus. This suggests that pSE228A likely functions as a stable environmental reservoir of virulence factors and heavy-metal resistance determinants within farm settings, thereby complementing the rapid dissemination capability of pSE228B. Collectively, pSE228A and pSE228B form a mobile, co-selective genetic platform that enhances the adaptability of SE JL228 under combined metal–antibiotic selective pressures and accelerates the emergence of hybrid pathogens, posing a substantial threat to both veterinary and public health.

The enhanced pathogenicity of SE JL228 is underpinned by a synergistic arsenal of plasmid-borne and chromosomal VFs. Notably, plasmid pSE228A encodes the complete Salmonella plasmid virulence (spv) operon (spvRABCD). The spv genes are established determinants for systemic disease, known to impair host immune responses [45, 46]. This genetic element provides a direct explanation for the markedly superior intracellular persistence of SE JL228 in APMs and its enhanced systemic infectivity compared to the spv-negative LN248 control strain. Critically, pSE228A also carries ibeB, an ortholog recognized for facilitating the invasion of hBMECs in pathogenic E.coli [47]. The presence of ibeB offers a strong functional interpretation for the neuroinvasive potential of SE JL228, correlating directly with the neurological symptoms (Additional file 2) and high bacterial loads detected in the brains of infected mice. Alongside these plasmid-mediated traits, the chromosome provides a robust virulence framework. The genome was confirmed to encode the complete Type III Secretion Systems (T3SS) SPI-1 and SPI-2. The SPI-1 T3SS and its effectors (e.g., sopB, sopE) are essential for host cell invasion, underpinning the high invasion rates observed in hBMECs, while the SPI-2 T3SS (e.g., sseC, sseD) is crucial for intracellular replication and survival within the phagosome, aligning with the APMs survival data [48, 49]. Furthermore, SE JL228 possesses key regulatory genes (csgD, adrA) and structural genes (csg, bcs) involved in biofilm formation, which play a crucial role in its environmental persistence. The resulting “rdar” morphotype provides physical protection against desiccation and disinfectants, while its adhesive properties facilitate colonization and dissemination within the farm environment [50]. This core framework is supported by additional chromosomal factors, including mgtC, which confers tolerance to magnesium limitation within macrophages [51]. The adhesion- and persistence-associated genes pagN, pagC, and pagD contribute to bacterial attachment to host cells, invasion of immune cells, and survival within the intracellular environment [52, 53]. Interestingly, despite the absence of several known virulence genes (e.g., orgB, sopD2, slrP), SE JL228 retained a potent virulent phenotype, suggesting that its comprehensive virulence repertoire contains functional redundancies or alternative compensatory mechanisms. It is this specific and complex constellation of VFs—combining core T3SS, biofilm capacity, the systemic spv operon, and the rare neuroinvasive ibeB gene—that defines the unique, hypervirulent profile of SE JL228, distinguishing it from typical regional strains and highlighting its significant public health risk. Future functional validation, such as the construction of isogenic spv and ibeB knockout mutants, is warranted to definitively dissect the specific contributions of these plasmid-borne factors to the hypervirulence and neuroinvasion observed.

The genomic architecture of SE JL228 does not represent an isolated evolutionary event but rather reflects the outcome of active interspecies gene exchange. Our comparative genomic analysis situates both plasmids within a broader epidemiological and ecological framework. The hybrid plasmid pSE228A shares a conserved backbone with pSE_AH228, confirming the widespread circulation of this IncHI2-type vector—well known for harboring both AMR and MRGs—among Salmonella isolates from agricultural environments [54]. Likewise, the high sequence homology between the IncN plasmid pSE228B and plasmids identified in Klebsiella pneumoniae indicates that JL228 efficiently acquires resistance determinants from a shared mobile gene pool circulating across diverse Enterobacteriaceae. The real concern emerging from these findings is the role of JL228 as an efficient “genetic sink.” This strain not only assimilated a K. pneumoniae-like IncN plasmid but also evolved or captured a hybrid IncHI2/IncFIB plasmid. Such a fusion is particularly alarming, as it combines the stable and promiscuous resistance backbone of IncHI2 with the classic virulence replicon spv-carrying IncFIB, revealing a potent mechanism for the co-selection and persistence of virulence and resistance traits under the intense environmental pressures of modern rabbit farming.

In conclusion, this study reports the identification of a hypervirulent, MDR SE strain isolated from a commercial rabbit farm, characterized by a hybrid conjugative plasmid that integrates virulence and resistance determinants. This discovery highlights a dual public health concern: the direct zoonotic risk of transmission through the rabbit meat supply chain and the broader threat of plasmid-mediated dissemination of resistance and virulence genes to other endemic pathogens. These findings call for a paradigm shift in food safety surveillance. It is recommended that the rabbit meat industry be incorporated into national monitoring frameworks, with traditional serotyping replaced by routine whole-genome sequencing (WGS)-based surveillance. Only through systematic genomic tracking of complex mobile genetic elements such as pSE228A, rather than focusing solely on bacterial species, can emerging antimicrobial resistance threats be anticipated and mitigated before escalating into major public health emergencies. While this study provides a detailed genomic and functional analysis of a single outbreak strain, further research involving diverse isolates from different regions and hosts is necessary to establish the broader epidemiological significance. Future investigations should explore the dissemination of IncFIB–IncHI2 and IncN plasmids in environmental and clinical contexts and elucidate how heavy metal resistance contributes to the persistence and spread of MDR SE, thereby enhancing our understanding of environmental influences on bacterial evolution.

Authors’ contributions

Writing-original draft: R.Z., H.Z. Writing-review and editing: H.Z. Investigation: R.Z., B.W., Y.L., J.L., X.D., H.Z. Validation: B.W., Y.L., X.D., J.Y., J.Z. Software: R.Z., B.W. Visualization: R.Z., B.W., H.Z. Formal analysis: J.Z., Y.L., J.Y., L.J. Data curation: Y.L., L.J., H.Z. Methodology: J.L., X.Y., J.Y., H.Z. Conceptualization: H.Z. Resources: J.Z., X.Y., J.Z. Funding acquisition: X.Y., H.Z, X.Z., Y.L. Supervision: L.J., H.Z., X.Z. Project administration: X.Z. All authors read and approved the final manuscript.

Funding

This work was supported by the Natural Science Foundation of Shandong Province, China (Grant No. ZR2024MC148 and ZR2023MC049), the Key Research and Development Plan of Shandong Province (Grant No. 2022CXPT022, 2025CXGC010803) and the Shandong Province Poultry Industry Technology System (Grant No. SDAIT-11–10).

Data availability

The complete genome sequence of Salmonella enterica serovar Enteritidis strain SEJL228A has been deposited in GenBank under accession number CP094269. The plasmid sequences are available under accession numbers CP094270 (pSE228A) and CP094271 (pSE228B). All data generated or analyzed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

Animals were handled according to the protocol reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ludong University (protocol No.: LDU-IACUC2019007).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.